Parallel advances in physics, instrumentation, and reporter technology have transformed biological imaging, such that it is now possible to visualize the localization, trafficking, interactions, and activity of many different proteins, with unprecedented spatial and temporal resolution inside living cells. However, a major challenge which affects the physiological relevance of all these experiments, has been largely un-addressed. Nearly all protein imaging experiments in living cells are performed using recombinant constructs, such as GFP, FlAsH, split ubiquitin, or luciferase fusions. Recombinant tags seriously affect biology in three ways: (1) tags can interfere with protein folding, trafficking, activity, and interactions; (2) protein over-expression leads to biological artifacts, such as changes in protein localization and interaction patterns; and (3) recombinant proteins do not have the same transcriptional and translational regulation as their endogenous counterparts, so these aspects of their biology cannot be easily studied. We propose to develop two technologies to enable the study of endogenous protein biology in living cells. First, we will develop a method to specifically and covalently label endogenous proteins of interest with small fluorophores and other probes inside living cells. This will be accomplished through in vitro evolution of two different small-molecule ligase enzymes ? biotin ligase and lipoic acid ligase ? to enable them to recognize and modify endogenous proteins. Second, we will develop a method to detect the endogenous interaction partners of specific cellular proteins. This method will be based on a promiscuous mutant of biotin ligase, which can be fused to proteins of interest, and which will release biotin-AMP ester that will covalently label any endogenous interacting partners. Biotinylated proteins can be purified and analyzed by mass spectrometry, after cell lysis. If successful, these two methodologies could transform biological studies of protein function in living cells, leading to fundamentally new discoveries of relevance to human health and disease.